Genomes are under constant assault from genotoxic and other agents which prevent accurate and efficient replication of DNA. Cells use many methods to continue replication past DNA lesions (known as damage tolerance). DNA damage tolerance may occur through an error-free or error-prone pathway. The error-free pathway of DNA damage tolerance prevents genome instability and chromosome breaks after DNA damage. Commitment to error-free damage tolerance hinges on polyubiquitin modification of the proliferating cell nuclear antigen (PCNA). PCNA is a homo-trimeric protein complex that encircles DNA, serving as a scaffold to enhance the processivity of DNA polymerases and coordinate other processes in DNA metabolism. In yeast (as in humans), polyubiquitination of PCNA by Rad5 with Ubc13'Mms2 initiates error-free damage tolerance, though the precise mechanism of initiation and bypass is unknown. Our goal is to characterize the structural and biochemical mechanisms of PCNA polyubiquitination and the role of Rad5 in this process. Polyubiquitination of PCNA is difficult to study using mutagenesis because it is a homo-trimer of the same gene product. Moreover, obtaining pure, homogenously monoubiquitinated PCNA from enzymatic methods is difficult. To circumvent these issues, we will construct vector that will express the homo-trimer of PCNA as a single protein, allowing mutagenesis and chemical ubiquitination. This protein will then, be used to identify the domain or domains of Rad5 important for binding and polyubiquitination of PCNA. Ultimately, the co-crystal structure of monoubiquitinated PCNA and Rad5 will be solved providing insight into this key process of genome maintenance. Defects in DNA damage tolerance in humans have recently been found to be significant to human health. Mutations and downregulation of HLTF and SHPRH, the human homologs of Rad5, are observed in colon, uterine, esophaegeal squamous cell carcinoma and other primary tumor cell lines. Ectopic expression of HLTF in colon cancer cells reduced tumor growth suggesting HLTF is a tumor suppressor protein. The functions of HLTF and SHPRH are likely linked to their role in polyubiquitination of PCNA and the initiation of error-free DNA damage tolerance.